JAK2/STAT3信号通路在阿托伐他汀抗大鼠血管平滑肌细胞增殖凋亡中的作用研究

目的探讨阿托伐他汀对大鼠血管平滑肌细胞(VSMCs)非调脂作用机制与转录信号传导子和激活子3(Stat3)信号传导通路的关系,明确其细胞内信号传导机制。方法分别将阿托伐他汀、酪氨酸激酶抑制剂AG490及二者联合作用于大鼠VSMCs,应用MTT法检测细胞增殖状态;流式细胞仪观察药物对细胞凋亡的影响,进一步用蛋白免疫印迹法(Western blot)检测药物作用前后 Stat3通路相关蛋白JAK2、STAT3、Cyclin D1、Bcl-2的表达及磷酸化活性。结果阿托伐他汀及AG490 都呈浓度依赖性方式抑制大鼠VSMCs增殖水平,促进其凋亡,二者联合应用则具协同作用。阿托伐他汀及AG490处理VSMCs后p-JAK2、p-Stat3、Cyclin D1、Bcl-2蛋白表达水平随作用时间延长而下降 (P<0．05),并且联合组蛋白表达水平较单独处理组相比有显著性差异(P<0．01)。结论阿托伐他汀对大鼠VSMCs非调脂作用机制与癌基因Star3信号通路高度相关,与酪氨酸激酶抑制剂AG490联合应用则具协同作用,可能通过作用于JAK2／Stat3下游靶基因Cyclin D1、Bcl-2影响其增殖及凋亡。     

反义EGFR寡核苷酸对AngⅡ促血管平滑肌细胞增殖效应的影响

目的探讨脂质体介导的反义表皮生长因子受体（EGFR）寡核苷酸基因转染对血管紧张素Ⅱ（AngⅡ）促大鼠血管平滑肌细胞（VSMC）增殖的影响。方法用EGFR寡核苷酸脂质体复合物转染Sprague-Dawley大鼠VSMC,通过RT-PCR、Western-Bloting分别检测转染后EGFR mRNA及蛋白的表达情况,用3H-Tdr掺入法检测经EGFR寡核苷酸转染再用AngⅡ刺激VSMC的增殖情况。结果反义EGFR寡核苷酸转染大鼠VSMC后,EGFR mRNA及蛋白的表达较正义组及对照组显著减少（P<0.01）;用AngⅡ刺激后,反义组细胞的3H-Tdr掺入较正义组及对照组显著降低（P<0.01）。结论脂质体介导的反义EGFR寡核苷酸转染可以减弱AngⅡ的促VSMC增殖效应。     

大鼠血管平滑肌细胞中以COX-2/JAK2/STAT3为靶向的信号转导分子调控机制的研究

目的:研究转录信号转导子和激活子3(STAT3)信号转导通路与选择性环氧化酶2(COX-2)抑制剂调控血管平滑肌细胞(VSMC)增殖凋亡的关系,明确COX-2抑制剂作用的细胞内信号转导机制。方法:将选择性COX-2抑制剂NS-398,作用于鼠VSMCs,运用MTT法检测细胞增殖状态;流式细胞仪观察NS-398对细胞凋亡的影响,进一步用RT-PCR检测药物作用前后VSMCs中COX-2mRNA表达;Westernblot检测药物作用前后STAT3通路相关蛋白JAK2、STAT3、CyclinD1、Bcl-2的表达及磷酸化活性。结果:鼠VSMCs中COX-2mRNA呈高表达,NS-398呈时间、剂量依赖性方式抑制VSMCs细胞增殖,促进其凋亡。加入NS-398的VSMCs中COX-2mRNA表达水平显著下降(P<0·01)。同时P-JAK2、P-STAT3、CyclinD1、Bcl-2表达水平随作用时间延长而下降(P<0·01)。结论:选择性COX-2抑制剂NS-398对VSMCs作用与COX-2mRNA水平相关,癌基因Stat3信号转导通路调控了NS-398对VSMCs作用的细胞内信号转导机制,最终通过其下游靶基因CyclinD1、Bcl-2影响VSMCs的增殖与凋亡。     

阻断p38信号转导通路对血管平滑肌细胞增殖和凋亡的影响

目的:研究老年大鼠血管平滑肌细胞（VSMCs）增殖和凋亡与p38信号转导通路的关系,明确以p38为靶向的信号转导在VSMCs中的分子调控机制。方法: 将p38特异性抑制剂SB203580(25 μmol/L)作用于大鼠VSMCs,运用MTT比色法检测细胞增殖状态,流式细胞术检测SB203580对细胞凋亡的影响,Western blot法检测药物作用前后p38通路相关蛋白p38α、MKK3、GADD153和c-myc的表达及相关蛋白磷酸化活性。结果: SB203580可以时间、剂量依赖的方式抑制VSMCs增殖促进其凋亡。加入SB203580的VSMCs中p-p38α、GADD153和c-myc表达的水平,随作用时间的延长而下降（P<0.01）。结论: 阻断p38信号转导通路可能通过下调其下游靶基因GADD153和C-myc的表达,抑制血管平滑肌细胞增殖,促进其凋亡。     

腺病毒介导反义AT1R转染对大鼠血管平滑肌细胞增殖的抑制作用

目的　观察腺病毒介导反义AT1基因转染对培养的大鼠动脉平滑肌细胞 (VSMCs)增殖的影响。方法　用定向克隆和同源重组方法构建携带人反义AT1基因的复制 -缺陷型重组腺病毒 (Ad/CMV·ahAT1) ,转染体外培养的VSMCs,用RT PCR半定量法和免疫组化法检测AT1R的表达 ,用3 H TdR掺入实验和流式细胞仪检测VSMCs的DNA合成和增殖指数。结果　与对照组相比 ,转染Ad/CMV·ahAT1后 4 8h的VSMCs,AT1R的mRNA表达低 5 0 % ,蛋白表达也显著低于对照组 (P <0 0 1)。给予AngⅡ刺激的VSMCs的3 H TdR掺入量和增殖指数明显高于空白对照组 (分别为P <0 0 5和P <0 0 1) ;转染Ad/CMV·ahAT1组3 H TdR掺入量和增殖指数则显著降低 (与DMEM组比P <0 0 5 ,与AngⅡ对照组比P <0 0 1)。 结论　腺病毒介导的反义AT1R转染 ,通过抑制AT1R的表达 ,明显抑制VSMCs的增殖和AngⅡ刺激的VSMCs增殖     

腺病毒介导反义AT1R转染对大鼠血管平滑肌细胞增殖的抑制作用

目的观察腺病毒介导反义AT1基因转染对培养的大鼠动脉平滑肌细胞(VSMCs)增殖的影响.方法用定向克隆和同源重组方法构建携带人反义AT1基因的复制-缺陷型重组腺病毒(Ad/CMV*ahAT1),转染体外培养的 VSMCs,用RT-PCR半定量法和免疫组化法检测AT1R的表达,用3H-TdR掺入实验和流式细胞仪检测VSMCs的DNA合成和增殖指数.结果与对照组相比,转染Ad/CMV*ahAT1后48 h的VSMCs,AT1R 的mRNA表达低50%,蛋白表达也显著低于对照组(P<0.01).给予Ang Ⅱ刺激的VSMCs的3H-TdR掺入量和增殖指数明显高于空白对照组(分别为P<0.05和P<0.01);转染Ad/CMV*ahAT1组3H-TdR掺入量和增殖指数则显著降低(与DMEM组比P<0.05,与Ang Ⅱ对照组比P<0.01).结论腺病毒介导的反义AT1R转染,通过抑制AT1R的表达,明显抑制VSMCs的增殖和Ang Ⅱ刺激的VSMCs增殖.     

VEGF对人血管平滑肌细胞株功能的影响及相关机制

目的探讨血管内皮生长因子(VEGF)对人血管平滑肌细胞(VSMCs)株增殖及凋亡的影响。方法体外培养的VSMCs细胞株采用0、0.1、0.2和0.4μg/ml的VEGF及0.4μg/ml的VEGF处理体外培养的VSMCs细胞株0、24、48、72 h,采用Western印迹方法检测VSMCs细胞株中磷脂酶C(PLC)-γ1信号通路的磷酸化活化情况,采用四甲基偶氮唑蓝比色法(MTT)和流式细胞仪(FCM)方法检测VSMCs细胞株增殖和凋亡能力的变化情况。采用PLC-γ1信号通路抑制剂U71322阻断VSMCs细胞株中PLC-γ1信号通路后,再以VEGF处理VSMCs细胞株,MTT和FCM方法检测VSMCs细胞株增殖和凋亡能力的变化情况。结果同0μg/ml的VEGF处理组相比,0.1、0.2、0.4μg/ml的VEGF可显著加强VSMCs细胞株的增殖能力(P0.01),并抑制VSMCs细胞株的凋亡水平(P0.01),且这种作用具有计量依赖性。Western印迹结果显示,0.1μg/ml的VEGF处理即可显著活化VSMCs细胞株中的PLC-γ1信号通路,且随着VEGF处理浓度的升高,PLC-γ1信号通路活化水平逐渐升高。当采用U71322阻断VSMCs细胞株中的PLC-γ1信号通路后,VEGF促VSMCs细胞株增殖的能力,以及抑制VSMCs细胞株的凋亡能力均显著降低(P0.01)。结论 VEGF可通过活化PLC-γ1信号通路,进而促进VSMCs细胞株的增殖能力,并抑制VSMCs细胞株的凋亡水平。     

Ap-1基因RNA干扰对大鼠血管平滑肌细胞增殖的影响

目的探讨Ap-1基因RNA干扰抑制血管平滑肌细胞（VSMCs）基因对VSMCs增殖的影响。方法以Ap-1基因RNA干扰VSMCs基因,应用MTr比色试验检测VSMCs增殖情况,流式细胞仪检测VSMCs细胞周期,免疫细胞化学染色观察增殖细胞核抗原（PCNA）表达。结果siRNA转染后,3个阳性siRNA转染组Ap-1基因表达水平均降低;VSMCs的MTY吸光度值和PCNA表达量均降低;VSMCs出现明显的G0／G1期阻滞。结论RNA干扰介导的Ap-1基因沉寂可显著抑制VSMCs增殖。     

氯通道ClC-3反义寡核苷酸对过氧化氢诱导的大鼠主动脉平滑肌细胞凋亡的影响

目的研究ClC3反义寡核苷酸对H2O2诱导的大鼠主动脉平滑肌细胞凋亡的影响。方法蛋白免疫印迹法检测ClC3蛋白表达;形态学方法、DNA琼脂糖电泳、MTT法和流式细胞仪观察和分析H2O2诱导的大鼠主动脉平滑肌细胞形态学改变、DNA断裂、细胞存活率和凋亡率及ClC3反义寡核苷酸转染对其影响。结果ClC3反义寡核苷酸转染抑制内源性ClC3蛋白表达后,可加重H2O2诱导大鼠主动脉平滑肌细胞形态学改变及DNA断裂,细胞凋亡率由52.8%±13.6%增至75.7%±5.8%(n=6,P<0.01),而细胞存活率由48.9%±4.3%进一步降低为31.3%±4.3%(n=6,P<0.01)。结论ClC3反义寡核苷酸转染促进H2O2诱导的大鼠主动脉平滑肌细胞凋亡。     

RNA干扰沉默Cyclin D1基因对胰腺癌AsPC-1细胞增殖和凋亡的影响

目的探讨以细胞周期蛋白（Cyclin）D1为靶基因的RNA干扰对胰腺癌AsPC-1细胞增殖和凋亡的影响,为胰腺癌的靶向治疗提供依据。方法用含有10％FBS的DMEM培养液在37℃、5％CO2培养箱中常规培养AsPC-1细胞,至对数生长期后接种于96孔培养板中,随机分为实验组、阴性对照组、空白对照组,并采用Lipo-fectamine^TM 2000脂质体分别转染Cyclin D1-小干扰RNA（siRNA）、阴性对照siRNA、脂质体,48h时收集细胞。应用荧光定量PCR法和Western blot法分别检测细胞中Cyclin D1 mRNA和蛋白表达水平,MTT法检测细胞体外增殖活力,流式细胞仪检测细胞周期分布及凋亡率（AI）。结果与空白对照组和阴性对照组相比,实验组Cyclin D1 mRNA和蛋白表达均明显下调,细胞生长速度明显减慢,G0／G1细胞比例显著增大、S期细胞比例则明显降低、AI显著升高（P均〈0．01）。结论Cyclin D1-siRNA能通过沉默靶基因表达抑制AsPC-1细胞生长、改变细胞周期分布、诱导细胞凋亡,可作为胰腺癌基因治疗的一个有效靶点。     

p38MAPK表达与血管平滑肌细胞增殖关系的研究

目的探讨丝裂素活化蛋白激酶p38(p38MAPK)的表达与血管平滑肌细胞(VSMC)增殖的关系以及检测p38MAPK反义寡核苷酸(AODN)对VSMC增殖的抑制作用。方法将培养大鼠胸主动脉VSMC,随机分为对照组、p38MAPKAODN组、正义寡核苷酸(SODN)组。采用噻唑蓝比色分析法(MTT)和流式细胞仪检测VSMC,用蛋白免疫印迹法测定p38MAPK蛋白量。结果p38MAPKAODN能减少p38MAPK蛋白表达,明显抑制VSMC增殖,其抑制作用与p38MAPK蛋白表达相关,呈剂量依赖性。结论p38MAPKAODN能抑制大鼠VSMC增殖,该信号分子与VSMC增殖密切相关,可能是VSMC增殖的信号途径。     

Deficiency in ClC-3 chloride channels prevents rat aortic smooth muscle cell proliferation

Recent growing evidence suggests that chloride (Cl-) channels are critical to the cell cycle. In cultured rat aortic vascular smooth muscle cells (VSMCs), we have previously found that Cl- channel blockers inhibit endothelin-1 (ET-1)-induced cell proliferation. The present study was designed to further identify the specific Cl- channels responsible for VSMC proliferation. Due to the lack of a specific blocker or opener of any known Cl- channels, we used the antisense strategy to investigate the potential role of ClC-3, a member of the voltage-gated Cl- channel gene family, in cell proliferation of cultured rat aortic VSMCs. With [3H]-thymidine incorporation and immunoblots, we found that ET-1-induced cell proliferation was parallel to a significant increase in the endogenous expression of ClC-3 protein. Transient transfection of rat aortic VSMCs with antisense oligonucleotide specific to ClC-3 caused an inhibition in ET-1-induced expression of ClC-3 protein and cell proliferation of VSMCs in the same concentration- and time-dependent pattern, whereas sense and missense oligonucleotides resulted in no effects on ClC-3 protein expression and cell proliferation. These results strongly suggest that ClC-3 may be the Cl- channel involved in VSMC proliferation and thus provide compelling molecular evidence linking a specific Cl- channel to cell proliferation. The full text of this article is available at http://www.circresaha.org.     

Stat5反义寡核苷酸联合5-氟尿嘧啶对胃癌细胞增殖与凋亡的影响

目的:探讨Stat5反义寡核苷酸(Stat5AS-ON)联合5-氟尿嘧啶(5-FU)治疗胃癌的作用机制.方法:Stat5AS-ON、5-FU单用或联用处理胃癌细胞BGC823,Westernblot检测Stat5、p-Stat5、cyclinD1与Bcl-xL表达,MTT法检测细胞增殖状态,流式细胞技术检测细胞周期与凋亡.结果:Stat5AS-ON与5-FU作用于BGC823细胞72h后,G1期细胞比率由65.7%上升至78.2%,S期细胞比率分别由18.6%,下降至10.5%,凋亡细胞百分比由7.4%增加至21.6%.Stat5AS-ON与5-FU可以抑制胃癌细胞增殖,促进胃癌细胞凋亡,联合应用Stat5AS-ON与5-FU可以起协同作用,明显抑制胃癌细胞Stat5信号转导通路活化.结论:选择性阻断细胞内信号转导通路可能为治疗胃癌提供新途径.     

Regulation of vascular smooth muscle cell apoptosis. Modulation of bad by a phosphatidylinositol 3-kinase-dependent pathway.

Our objective was to define the signaling mechanisms by which mitogens such as insulin-like growth factor-I (IGF-I) regulate vascular smooth muscle cell (VSMC) apoptosis. We confirmed that IGF-I inhibits serum withdrawal-induced apoptosis of cultured VSMCs in a dose-dependent and time-dependent fashion. To test the hypothesis that the phosphatidylinositol (PI) 3-kinase signaling pathway regulates VSMC survival, we examined the relationship between PI 3-kinase activity and cell fate. PI 3-kinase was elevated in viable VSMCs maintained in serum-containing medium, declined significantly in response to serum withdrawal, and increased in response to IGF-I-induced survival. Moreover, blockade of PI 3-kinase with 2 structurally dissimilar inhibitors (wortmannin or LY294002) abolished the capacity of IGF-I to maintain VSMC viability. Similarly, transient transfection of a dominant-negative Deltap85 PI 3-kinase mutant construct abrogated the capacity of IGF-I to prevent VSMC death. Thus, PI 3-kinase is a critical antiapoptotic signal in VSMCs. To define the distal element of the antiapoptotic cascade, we tested the hypothesis that IGF-I inhibits the influence of the proapoptotic gene Bad. Indeed, IGF-I stimulates increased expression of the inactive, phosphorylated form of Bad by a PI 3-kinase-dependent pathway. Moreover, the proapoptotic effect of Bad was attenuated by the stimulation of IGF-I. Thus, growth factors appear to prevent VSMC death by activating signal transduction pathways linked to apoptotic regulatory genes.     

FK506 can activate transforming growth factor-beta signalling in vascular smooth muscle cells and promote proliferation

AIMS: FK506-binding protein (FKBP) 12 is an inhibitor of transforming growth factor (TGF)-beta type I receptors. Several lines of evidence support the view that TGF-beta stimulates vascular smooth muscle cell (VSMC) proliferation and matrix accumulation. We investigated the effect of FK506, also known as tacrolimus, on cellular proliferation and on matrix protein production in human VSMCs. METHODS AND RESULTS: We measured cell proliferation with flow cytometry using BrdU incorporation and fluorimetrically by measuring DNA concentration with Hoechst 33258. Western blot assay of whole-cell lysates was used to measure the levels of signalling proteins involved in proliferative pathways, in particular beta-catenin, pErk, pAkt, pmTOR, and cyclin D1. Collagen synthesis was also investigated by Western blotting. The TGF-beta signal was studied by both Western blotting and confocal microscopy. We used the SiRNA technique for FKBP12 gene silencing. Our results show that FK506 stimulates VSMC proliferation and collagen type I production. FK506 enhanced beta-catenin levels and activated the extracellular signal-regulated kinase, Akt, and mammalian target of rapamycin kinase, which are important effectors of proliferation. Accordingly, cyclin D1 expression was increased. We also demonstrate that FK506 activates the TGF-beta signal in VSMCs and that, through this mechanism, it stimulates cell proliferation. CONCLUSION: FK506 can act as a growth factor for VSMCs.     

Kallistatin stimulates vascular smooth muscle cell proliferation and migration in vitro and neointima formation in balloon-injured rat artery

Kallistatin, a serine proteinase inhibitor (serpin), is expressed in the endothelial and smooth muscle cells of blood vessels. The potential function of kallistatin in vascular biology was investigated by studying its role in the proliferation and migration of cultured primary aortic vascular smooth muscle cells (VSMCs) in vitro and in neointima formation in rat artery after balloon angioplasty in vivo. Exogenous kallistatin induced a >2-fold increase of VSMC proliferation and cell growth as measured by [(3)H]thymidine incorporation and cell counts and a 2.3-fold increase of cell migration in modified Boyden chambers. In balloon-injured vessels, endogenous kallistatin mRNA and protein levels increased up to 10-fold as determined by competitive polymerase chain reaction and by ELISA. Intense staining of kallistatin mRNA was identified in the proliferating VSMCs of balloon-injured arteries during cell migration from media to neointima by in situ hybridization histochemistry and immunohistochemistry. We observed an induction of kallistatin expression by platelet-derived growth factor (PDGF) and upregulation of p42/44 mitogen-activated protein kinase (MAPK) activity by kallistatin in cultured VSMCs. Conversely, adenovirus-mediated transfer of kallistatin antisense cDNA into cultured VSMCs inhibited PDGF-induced p42/44 MAPK activity and cell proliferation. Furthermore, local delivery of adenovirus carrying kallistatin antisense cDNA significantly downregulated kallistatin mRNA levels and attenuated neointima formation in balloon-injured rat arteries in vivo. These results indicate that kallistatin may play an important role in mediating PDGF-induced MAPK pathway on VSMC proliferation and in neointima formation after balloon angioplasty.